National foreword

This British Standard is the UK implementation of ISO 4962:2024.

The UK participation in its preparation was entrusted to Technical Committee NTI/1, Nanotechnologies.

A list of organizations represented on this committee can be obtained on request to its committee manager.

Contractual and legal considerations

This publication has been prepared in good faith, however no representation, warranty, assurance or undertaking (express or implied) is or will be made, and no responsibility or liability is or will be accepted by BSI in relation to the adequacy, accuracy, completeness or reasonableness of this publication. All and any such responsibility and liability is expressly disclaimed to the full extent permitted by the law.

This publication is provided as is, and is to be used at the recipient’s own risk.

The recipient is advised to consider seeking professional guidance with respect to its use of this publication.

This publication is not intended to constitute a contract. Users are responsible for its correct application.

Compliance with a British Standard cannot confer immunity from legal obligations.

This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 November 2024.

Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types of ISO document should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

ISO draws attention to the possibility that the implementation of this document may involve the use of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s) which may be required to implement this document. However, implementers are cautioned that this may not represent the latest information, which may be obtained from the patent database available at www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.

Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.

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This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.

Any feedback or questions on this document should be directed to the user’s national standards body. A complete listing of these bodies can be found at www .iso .org/members .html.

Introduction

Phototoxicity (PT) is defined as a toxic response to an organism exposed to a substance, where the response is either elicited or increased (apparent at lower dose levels) after subsequent exposure to light, or that is induced by skin irradiation after systemic administration of a substance. The increasing use of nanomaterials in various industries also leads to increased exposure, especially to skin. Furthermore, some nanomaterials are used in commercial sunscreens. Hence, possible impacts on human health including detrimental chemical reactions in the presence of light (both natural and artificial) or photo-protective effects, is of interest.

PT is based on a quantum phenomenon. The absorption of a photon with sufficient energy generates an electron-hole pair that can migrate to the nanoparticle (NP) surface and react with water and oxygen, thus forming extremely reactive radicals and reactive oxygen species (ROS). Generation of the ROS by some wide-bandgap materials, such as TiO₂, ZnO, WO₃, CeO₂, carbon nanotubes, quantum dots and some metal NPs when illuminated by UV-VIS light, can cause oxidative stress, resulting in toxic effects in living organisms. Absorption of a photon with sufficient energy is the necessary condition for photochemical reactions to induce phototoxic response. Material PT is closely related to photocatalytic activity (PCA). Measurement of PCA under physiological conditions allows for an assessment of its phototoxicity potency (see ISO 20814).

A wide variety of light sources, light exposure levels, cell lines, incubation times, viability assays, used for nanomaterial PT measurement hamper data comparison. Existing PT standard test methods for soluble chemical substances (e.g. OECD 432) are not directly applicable to determine nanomaterial PT. It states that DMSO or EtOH should be used as cosolvents in case the material is not soluble in water with additional tests for cosolvent toxicity. The method is not applicable for particulate materials.

The in vitro NP PT test is intended to evaluate the nanomaterial acute phototoxicity when exposed to a near ultraviolet (UVA) light. Cell viability is assessed at a fixed NP concentration after exposure to six doses of the UVA light.

1 Scope

This document specifies a procedure to evaluate acute phototoxicity of nanoparticles (NPs), suspended in cell culture media, by measuring the relative reduction in cellular viability under near ultraviolet (UVA) exposure (315 nm to 400 nm).

The measurement is intended to assess the potential for acute phototoxicity of NPs by comparing NP photoactivity in vitro to a positive control chlorpromazine. It is not designed to predict other joint effects of nanomaterials and light, such as genotoxicity, photo-allergy or photo-mutagenicity. The method is also applicable to NP aggregates and agglomerates.

2 Normative references

3 Terms, definitions, symbols and abbreviated terms

3.1 Terms and definitions

3.2 Abbreviated terms

4 Test method

5 Materials and equipment

5.1 Materials

5.2 Apparatus

6 Sample preparation

7 Preparations

7.1 General

7.2 Culture medium

7.3 Preparation of cell stock culture

7.4 Verify viable cell growth

7.5 Irradiation conditions

7.6 Multiple well plate reader

8 Measurement procedure

8.1 Cell seeding (Day 1)

8.2 Incubation of cells with the positive control and NP suspension (Day 2)

8.3 UVA exposure (Day 3)

8.4 Cell viability assay (Day 4)

8.5 Evaluation of artefacts due to possible NP interferences with the MTS assay (in the dark)

8.6 Data analysis

9 Report

9.1 Test report

9.2 Report data format

10 Precision

10.1 Repeatability

10.2 Reproducibility

Bibliography

BS ISO 4962:2024

Nanotechnologies. In vitro acute nanoparticle phototoxicity assay

Current

Published:

30 Nov 2024